A brainstem circuit for nausea suppression

SUMMARY Nausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects blood-borne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through γ-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention.


INTRODUCTION
Nausea is one of the most encountered symptoms in healthcare, with current anti-nausea medications displaying variable clinical success (Hesketh, 2008). New strategies for nausea intervention are needed and may be enabled by a mechanistic understanding of how the sensation of nausea arises. Classical studies involving brain lesion and stimulation revealed a tiny brainstem structure termed the area postrema that mediates nausea responses to several visceral threats (Borison, 1989). The area postrema is a sensory circumventricular organ with a privileged anatomical location containing a reduced blood-brain barrier, which allows resident neurons to sample hormones and other chemicals in the circulatory system (Borison, 1989).
Single-cell cDNA sequencing approaches recently provided a cell atlas of the area postrema, revealing four excitatory and three inhibitory neuron types (Zhang et al., 2021). One excitatory neuron type expresses multiple receptors for nausea-inducing stimuli, including the growth/differentiation factor 15 (GDF15) receptor (GFRAL), the glucagon-like peptide 1 (GLP1) receptor (GLP1R), and the calcium-sensing receptor (CaSR). GFRAL, GLP1R, and CaSR agonists cause nausea and/or vomiting in large animals and in mice, which are incapable of vomiting, evoke a characteristic behavioral response termed conditioned flavor avoidance in which paired administration of a poison and a novel flavor causes future avoidance of that flavor (Andrews, 1992;Patel et al., 2019;Zhang et al., 2021). Chemogenetic activation of area postrema GFRAL neurons promotes flavor avoidance, while their ablation eliminates avoidance imposed by several visceral poisons (Sabatini et al., 2021;Zhang et al., 2021). Thus, area postrema GFRAL neurons are a key node in nausea circuits, and inhibitory pathways that lower their activity may reduce symptoms of gut malaise.
The functions of area postrema inhibitory neurons are unclear. All three area postrema inhibitory neuron types (and none of the excitatory neurons) are marked in Gad2-ires-Cre mice, and genetic tracing previously revealed their projections to be local and largely confined to the area postrema, with minor projections observed in the adjacent nucleus of the solitary tract (NTS) and not in other brain regions that receive excitatory area postrema inputs (Zhang et al., 2021). Based on this observation, we hypothesized that at least some inhibitory neuron types may suppress the activity and function of area postrema excitatory neurons, including those involved in nausea.

Area postrema inhibitory neurons suppress local excitatory neurons and poison responses
We used channelrhodopsin (ChR2)-assisted circuit mapping (CRACM) to investigate the connectivity patterns of area postrema inhibitory neurons. Gad2-ires-Cre, Rosa26lsl-L10GFP mice were injected in the area postrema with an AAV containing a Credependent ChR2-mCherry allele (AAV-Flex-ChR2-mCherry); post hoc histological analysis confirmed that viral infection was largely restricted to the area postrema (Figures 1A and 1B). Whole-cell recordings in area postrema tissue slices revealed robust light-gated currents in mCherry-positive inhibitory neurons ( Figure 1C). Post-synaptic responses were then measured in area postrema excitatory neurons, which were identified by neuronal morphology and a lack of GFP fluorescence in Gad2-ires-Cre, Rosa26-lsl-L10GFP mice.
Optogenetic activation of area postrema inhibitory neurons produced large outward chloride currents (using high-chloride intracellular solution) in area postrema excitatory neurons (resting membrane potential: −49.3 ± 1.1 mV; Figure S1A) that were abolished by application of the GABA A receptor antagonist bicuculline ( Figure 1D). Light-evoked inhibitory post-synaptic currents (IPSCs) in most excitatory neurons (89%, 16/18) displayed onset kinetics and insensitivity to tetrodotoxin ( Figures 1D and S1B), consistent with a direct monosynaptic connection from inhibitory neurons. Post-synaptic responses were also observed with reduced frequency in 27% (6/22) of area postrema inhibitory neurons (mCherry-negative, GFP-positive neurons) and 38% (8/21) of excitatory neurons in adjacent NTS regions (Figures S1C and S1D). Together, these findings indicate that most area postrema excitatory neurons receive local inhibitory input, with area postrema inhibitory neurons also forming functional connections with some NTS excitatory neurons and other area postrema inhibitory neurons.
We activated area postrema inhibitory neurons using chemogenetic approaches and observed the consequences on mouse behavior. We injected Gad2-ires-Cre mice in the area postrema with or without adeno-associated viruses (AAVs) containing Cre-dependent genes encoding designer Gα s -coupled receptors (AAV-Flex-GsDREADD-mCherry) activated by the synthetic agonist clozapine-N-oxide (CNO) (Roth, 2016); Gα s -coupled receptors were used based on commonly expressed area postrema receptors (see below). Chemogenetic activation of area postrema inhibitory neurons was validated by measuring CNO-induced Fos expression in mCherry-labeled neurons ( Figure S2A). Proper AAV targeting of the area postrema was confirmed in every animal post hoc by blinded histological analysis of mCherry expression; we sometimes observed targeting of a few nearby NTS neurons ( Figure  S2B), and animals lacking labeled area postrema neurons were excluded from analysis (see STAR Methods for more information).
We used an established behavioral paradigm to measure conditioned flavor avoidance (Patel et al., 2019;Zhang et al., 2021). Briefly, on a conditioning day, water-restricted mice were given access to a novel flavored saccharin solution (either cherry or grape flavored) and then immediately injected with saline (control), poisons, and/or CNO. Subsequently, on a testing day, behavioral preference for cherry-or grape-flavored solution was measured using a two-choice assay and expressed as a preference index where the time drinking the conditioned flavor was divided by total time drinking. In the absence of malaise induction, mice displayed a modest preference for the experienced flavor, with similar results observed for cherry and grape (Zhang et al., 2021). In contrast, robust behavioral avoidance of the experienced flavor was evoked by various poisons (Zhang et al., 2021), as well as the GFRAL agonist GDF15, as reported previously (Patel et al., 2019). CNO-induced activation of area postrema inhibitory neurons did not evoke flavor aversion or attraction in this paradigm in the absence of poison induction ( Figure 1E). Strikingly, however, when chemogenetic activation of area postrema inhibitory neurons was paired with GDF15 or lithium chloride injection, imposed aversion was lost ( Figure 1E). CNO silenced GDF15 and lithium chloride responses in mice with DREADD expression in inhibitory neurons but had no effect in control mice lacking DREADD expression, either in the presence or absence of poisons. Together with anatomical and connectivity-mapping studies, these findings indicate that at least some area postrema inhibitory neurons project to and inhibit the activity and function of nausea-promoting excitatory neurons.

GIP inhibits GFRAL neurons through a monosynaptic connection
We focused on one subpopulation of area postrema inhibitory neurons that express the receptor (GIPR) for glucose insulinotropic peptide (GIP). GIP is a gut-derived hormone and incretin released upon nutrient intake that rapidly promotes insulin release (Baggio and Drucker, 2007). Small molecules that activate the receptor for another incretin, GLP1, are clinical mainstays for diabetes treatment but induce nausea as an adverse side effect through area postrema excitatory neurons (Drucker and Nauck, 2006;Zhang et al., 2021). Recent studies involving paired administration of both incretins-GIP and GLP1-observed that GIP suppressed some adverse behavioral responses to GLP1 (Borner et al., 2021); furthermore, GIPR agonists reduced morphine-and cancer-drug-induced vomiting in ferrets (Asami et al., 2018). The neuronal basis for GIP responses remains unclear, with several gut-brain communication routes possible.
We previously revealed by single-cell cDNA sequencing that Gipr is expressed in a subset of area postrema inhibitory neurons (neuron cluster 6; Figure 2A) (Zhang et al., 2021). Since we observed here that area postrema inhibitory neurons suppress poison responses, we hypothesized that cluster 6 neurons may directly mediate or contribute to the anti-nausea effects of GIP and that targeting these neurons could represent a general strategy for nausea intervention. Moreover, GIPR expression provides a selective molecular handle for both genetic and pharmacological control of cluster 6 neurons. We obtained Gipr-Cre mice (Adriaenssens et al., 2019) and validated efficient targeting of cluster 6 area postrema neurons by two-color expression analysis involving dual visualization of a Cre-driven fluorescent reporter and Gipr mRNA by in situ hybridization ( Figure S3A); as is common with Cre tools, we noted some reporter-positive, Gipr-negative cells, more so in the NTS than the area postrema, which may have resulted from Gipr expression that is transient or too low for detection by RNA in situ hybridization.
We asked whether GIP evoked responses in area postrema cluster 6 neurons marked in Gipr-Cre, R26-lsl-tdTomato mice; as a note, we used [D-Ala 2 ]-GIP as a stable GIP analog in all experiments. GIPR is a Gα s -coupled receptor, and we observed GIP-evoked cAMP transients in dissociated GIPR neurons (71.4%, 15/21 cells) using the genetically encoded fluorescent cAMP sensor cADDis ( Figure 2B) (Tewson et al., 2016). Increasing cAMP levels depolarizes some, but not all, neurons, so we asked whether GIP also evokes electrical responses in cluster 6 neurons. Whole-cell, patch-clamp analysis in area postrema slices revealed robust GIP-evoked depolarization and action potentials in tdTomato-positive neurons (9/17 cells) from Gipr-Cre, R26-lsl-tdTomato mice ( Figures 2C and 2D). For comparison, GIP rarely depolarized (2/74 cells) or evoked cAMP transients (3/29 cells) in other area postrema neurons lacking tdTomato expression; instead, hyperpolarization of some tdTomato-negative neurons was observed (23%, 17/74 neurons). Intraperitoneal (i.p.) injection of GIP also induced Fos expression in area postrema neurons, with effects persisting after vagotomy, suggesting that vagal inputs were not required for area postrema responses to GIP ( Figure S3B). Taken together, GIP evokes characteristic and direct responses in GIPR-expressing area postrema inhibitory neurons that include increased cAMP levels, depolarization, and cell firing.
We used genetic approaches in Gipr-Cre mice to map the anatomy of area postrema cluster 6 neurons and, for comparison, used Glp1r-ires-Cre mice to map area postrema excitatory neuron clusters 2-4 ( Figure 2E) (Williams et al., 2016;Zhang et al., 2021). We injected AAV-Flex-GFP and AAV-Flex-Synaptophysin-mCherry into the area postrema to label neuronal cell bodies and synaptic terminals, respectively. As we observed previously, area postrema excitatory neurons project to multiple brain regions including the NTS, parabrachial nucleus (PBN), and autonomic motor nuclei (Zhang et al., 2021). In contrast, we observed that area postrema GIPR neurons, like inhibitory neurons in bulk, displayed dense arborizations within the area postrema and minor projections to proximal NTS, but GIPR neuron projections were not observed in the PBN or autonomic motor nuclei. These findings raised the possibility that GIPR neurons form inhibitory contacts with some or all area postrema excitatory neurons.

GIP suppresses nausea behaviors through area postrema inhibitory circuits
GFRAL neurons mediate nausea-associated responses to several visceral threats (Zhang et al., 2021), so we reasoned that inhibiting GFRAL neurons through local area postrema circuits may provide an effective way to attenuate poison responses. Pharmacological access to cluster 6 neurons is facilitated as the area postrema contains a reduced blood-brain barrier and local neurons can directly detect circulating peptides like GIP. We tested whether GIP could suppress nausea-related behaviors and whether GIP-evoked behavioral responses required area postrema inhibitory circuits. Using the conditioned-flavor-avoidance paradigm described above, wild-type mice were administered either GIP or saline on the conditioning day and 20 min later were exposed to GDF15 or LiCl. GDF15 and LiCl evoked robust flavor-avoidance responses in saline-injected control mice, but responses were abolished in mice receiving a prophylactic GIP injection ( Figure 4A).
We asked whether area postrema cluster 6 neurons were required for the suppression of nausea-related behavior by GIP. We used a genetic approach involving diphtheria toxin (DT) to ablate Cre-expressing GIPR neurons in the area postrema. Mouse cells are DT resistant but can be made susceptible by Cre-guided expression of the DT receptor (DTR) (Buch et al., 2005). DT was injected into the area postrema of Gipr-Cre; Rosa26-lsl-DTR mice (Gipr-ABLATE AP mice) or of Cre-negative Rosa26-lsl-DTR mice (non-ABLATE mice) as a control. Gipr-ABLATE AP mice displayed near-complete loss of cluster 6 neurons, but not other area postrema neuron types, and some loss of GIPR neurons in the NTS but not loss of GIPR neurons in other brain regions such as the hypothalamus ( Figures  4B and 4C). Behavioral responses of Gipr-ABLATE AP mice and non-ABLATE mice were examined using the conditioned-flavor-avoidance paradigm. We observed that GIP protected against GDF15-conditioned flavor avoidance in non-ABLATE mice but no longer suppressed GDF15 responses in Gipr-ABLA-TE AP mice ( Figure 4D). Thus, GIP suppresses nausea-associated behavior through area postrema inhibitory neurons.

DISCUSSION
Nausea evoked by visceral poisons can be counterproductive, causing patients to forego life-saving medications for cancer, diabetes, and other diseases. Studies here establish area postrema inhibitory neurons (cluster 6, GIPR neurons) as a target for suppressing behavioral responses to at least some nausea-inducing toxins ( Figure 4E). GIP is a gut hormone and incretin that activates GIPR neurons, and GIPR neurons in turn suppress the activity of nearby GFRAL-expressing and nausea-promoting excitatory neurons. In addition to GIPR, cluster 6 neurons express other cell surface receptors (Zhang et al., 2021), including mu-opioid receptor and neuropeptide Y receptor 2; it is possible that activating these receptors stimulates or inhibits GIPR neurons, with other stimulating agonists potentially providing additional avenues for pharmacological modulation of nausea-related behaviors with variable associated side effects. Furthermore, the strategy of targeting inhibitory circuits will likely be applicable to other area postrema neurons, including SLC6A2 neurons, which also condition flavor avoidance (Zhang et al., 2021) and receive distinct local inhibitory input from GAD2-positive, GIPR-negative neurons. Other inhibitory neurons (cluster 5) express the ghrelin receptor (Zhang et al., 2021), raising the possibility that several gut hormones act in concert to toggle the balance of area postrema inhibition and excitation. GIP is released following nutrient-rich meals (Baggio and Drucker, 2007), and it may be beneficial under certain physiological conditions for animals to consume calorie-rich foods containing small amounts of harmful chemicals. Area postrema circuits that integrate reward and punishment signals could guide future consummatory decisions based on need, reward value, and toxin risk. Together, experiments here reveal that GIP suppresses poison responses through a dedicated neuronal pathway, key insights into area postrema circuit organization, and a potential strategy for nausea intervention that involves targeting of brainstem inhibitory neurons.

Limitations of the study
GIP directly stimulates cluster 6 neurons in slice preparations ( Figure 2B), and peripheral GIP injection induces area postrema Fos expression ( Figure S3B), but it has not been demonstrated that a sufficient concentration of meal-induced GIP will naturally arrive at the brainstem to stimulate area-postrema-localized GIPR. Area postrema GIPR neurons are required for GIP responses reported here ( Figure 4D), yet a less parsimonious model is that GIP acts through an upstream neuronal route to engage area postrema GIPR neurons indirectly. GIP does not activate GIPR-negative area postrema neurons ( Figures 2D and 3), and while the area postrema receives predominant input from the vagus nerve, area postrema GIP responses persist in vagotomized mice ( Figure S3B). Additional studies are needed to understand when GIPR neurons are naturally engaged and whether they are activated by meal consumption, vagal inputs, top-down inputs, and/or other stimuli.

STAR★METHODS RESOURCE AVAILABILITY
Lead contact-Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Stephen Liberles (Stephen_liberles@hms.harvard.edu).
Materials availability-All mouse lines are available as previously described (Zhang et al., 2021), except Gipr-Cre which is available from Frank Reimann upon reasonable request.
• This paper does not report original code.
• Any additional information related to data reported is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
All animal husbandry and procedures were performed in compliance with institutional animal care and use committee guidelines. All animal husbandry and procedures followed the ethical guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals (https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-oflaboratory-animals.pdf), and all protocols were approved by the institutional animal care and use committee ( (007900) mice were purchased (Jackson Laboratory). Both male and female mice between 8-24 weeks old were used for all other studies, and no differences based on sex were observed.

METHOD DETAILS
Electrophysiology and circuit mapping-Brain tissue was dissected from mice (6-10 weeks of age) following anesthesia (isoflurane) and decapitation, and brain slices (200 μm Photostimulation-evoked IPSCs were recorded in the whole-cell, voltage-clamp mode, with membrane potential clamped at −60 mV. To photo-stimulate channelrhodopsin-positive cells, an LED light source (473 nm; CoolLED, Andover, UK) was used and controlled by the pClamp 10.2 software (Axon Instruments), with a photostimulation involving five 50 ms blue light laser pulses administered 1 s apart. In some experiments, 10 μM bicuculline methiodide (Tocris 2503)  , and for display, smoothened with the smoothdata function, as a moving average with an automated window length. The baseline activity for each neuron was defined as the average green fluorescence intensity over a five-minute period preceding stimulus delivery, and cells were excluded if they failed to display responses to positive controls.
Analysis of RNA, protein, and reporter expression-Hybridization chain reaction (HCR) RNA in situ hybridization was performed on cryosections of unfixed brain (25 μm) following the HCR-FISH 3.0 protocol (Choi et al., 2018(Choi et al., , 2020. Gipr probe, probe hybridization buffer, probe wash buffer, amplification buffer, and fluorescent HCR hairpins were purchased from Molecular Instruments (Los Angeles, USA). Gipr probe was designed to be associated with the B3 initiator sequence and detected by hairpins labeled with Alexa Fluor 647 (Lot# PRL060). Fluorescent images were analyzed with a Leica SP5 II confocal microscope.
Behavioral assays-Conditioned flavor avoidance assays involved a seven-day protocol with daily 30-minute introductions to a test arena containing two water bottles soon after dark onset. Mice had no access to water in the home arena but given ad libitum water access in the test arena. In the first three days (habituation days), both water bottles contained unflavored water. On day four (conditioning day), both water bottles were filled with either grape-flavored or cherry-flavored water (grape or cherry Kool-Aid) sweetened with 0.2% saccharin, each on half of the trials. Immediately after test arena occupancy on the conditioning day, mice were injected with saline (10 μL/g, IP) alone or containing either CNO (1 mg/kg, Tocris 6329), GDF15 (20 μg/kg, R&D systems 957-GD-025/CF),  ]-GIP (374 μg/kg), or LiCl (168 mg/kg). On day five (recovery day), both water bottles contained unflavored water. On day six (testing day 1), one water bottle contained cherry-flavored water, while the other contained grape-flavored water on a randomized arena side, and consumption from each water bottle scored using an automated lickometer based on a published design (Slotnick, 2009). On day seven (testing day 2), the positions of the two bottles containing either grape-or cherry-flavored water were switched. Preference index was calculated as time drinking the conditioned flavor water divided by total time drinking and was based on the first test day results.
In neuronal imaging experiments, cells were counted as responsive if stimulus-evoked ΔF/F exceeded three standard deviations above the baseline mean. Statistical significance was measured on Prism 9 software (Graphpad) using a Mann-Whitney test ( Figures 1E, 4A and 4D) and a Mann-Whitney test with Bonferroni correction ( Figure 4C).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.